PROJECT SUMMARY/ABSTRACT Asthma is one of the most common chronic respiratory disorders, estimated to affect 1 in 13 people in the U.S. Although advances in treatment have significantly improved disease management, poorly controlled asthma is still associated with significant morbidity and mortality. The current strategy to improve asthma control focuses on reducing inflammation. GATA3, a transcriptional activator, is involved in T lymphocyte differentiation and signaling, particularly in the Th2 subtype, and regulates the expression of cytokines such as IL-4, IL-5, and IL- 13, which play major roles in the inflammation responsible for asthma. Accordingly, knock down of GATA3 expression is a promising strategy for treating asthmatic patients with the Th2 endotype. A range of antisense and RNAi technologies have been tested, and among these approaches, DNA enzymes (Dzs) have shown the greatest promise in animal models and Phase 1 clinical trials. Dzs are canonical DNA oligonucleotides that catalytically degrade a specific complementary RNA sequence. Despite the success of soluble Dzs as a therapeutic intervention, delivering highly charged oligonucleotides across the plasma membrane, and preventing nuclease degradation are major challenges. To address these problems, we propose developing GATA3 DNAzyme nanoparticle conjugates that elucidate the stability and delivery issues in the lung. Preliminary evidence shows that conjugating ~100 Dzs to a 14-nm gold particle forms a complex (DzNP) that improves airway function in mouse models of asthma. Importantly, DzNPs use one order of magnitude lower Dz dose compared to their soluble counterparts. A fundamental question pertains to how DzNPs mediate improved efficacy. The central hypothesis is that DzNPs differentially target resident cell types that overexpress class A scavenger receptors, which are primary contributors to the Th2 asthma subtype. To test this hypothesis, I propose the following specific aims: Aim 1 will measure scavenger receptor A expression levels, DzNP uptake, and GATA3 knockdown efficiency in lung cell lines. The goal is understanding how DzNP treatment differs from that of Dzs and whether the cell targets mediate improved efficacy. Aim 2 will determine whether the DzNP acts as a delivery vehicle for Dz payloads, or whether the DzNP construct is the functional agent mediating GATA3 regulation. This will be achieved by employing fluorescence lifetime imaging (FLIM) microscopy probes that report the molecular environment of the Dz molecule. The trainee will master a wide range of engineering and biological techniques, including nanoparticle design, characterization, development and microscopy. The proposed research will provide insight into a novel method of gene regulation using a synthetic biomaterial. This research will provide a foundation for future development of nanoparticle-based therapeutic strategies for numerous diseases.